The long term goals of this study are to define the changes in ion channel activity in colonic inflammation. Ulcerative colitis is an inflammatory bowel disease characterized by recurrent episodes of colonic inflammation and tissue degeneration. The overall hypothesis is that inflammation induces specific changes in expression, regulation and kinetics of ion channels in smooth muscle cells. These changes affect gastrointestinal motility contributing to the clinical expression of altered contractions. In this proposal, we will test the hypothesis that reactive nitrogen species produced during colonic inflammation cause nitration of the tyrosine residues within the c-terminus of the calcium channel and result in down-regulation of channel function. Based on our preliminary findings, our working model is that nitration of tyrosine residues within the c-terminus of the Ca2+ channel prevents phosphorylation by c-src kinase and alters channel gating, downstream intracellular signaling and gene transcription.
Specific aim 1 is to determine the mechanisms by which c-src kinase alters the gating properties of the smooth muscle calcium channel. In this aim, we will determine the biophysical properties of the calcium channel and examine the effect of tyrosine phosphorylation and nitration on voltage-dependent inactivation using single channel and whole-cell patch clamp techniques. We will also investigate the interaction of src kinase with the calcium channel using fluorescence resonance energy transfer (FRET) and siRNA approaches to delineate the functional effect of src-Ca2+ channel interaction in muscle contraction. Preliminary data show that mutation of the terminal tyrosine residue within the c-terminus of the Ca2+ channel prevents the docking of the src homology 2 (SH2) domain.
In specific aim 2 we will examine the role of nitration of the Ca2+ channel in intracellular signaling and gene expression following inflammation. In this aim we will measure phosphorylation of the transcription factor CREB, and the downstream activation of cyclic AMP response element (CRE). We will test the effect of Ca2+ channel nitration on these processes and define the role of the terminal tyrosine residues using mutants of the Ca2+ channel. Analogous to protein phosphorylation-dephosphorylation, we will test whether denitration is a physiological process in specific aim 3. Our preliminary findings indicate that Ca2+ channel are substrate(s) for denitration. We will test whether denitration reverses the down-regulation of calcium influx in whole tissue segments, and determine the source of denitrating enzymes within the colonic wall. The information obtained from these studies will increase our understanding of the potential changes in ion channel activity with inflammation and help identify novel therapeutic agents in the treatment of motility disturbances in the pathophysiology of the colon.

Public Health Relevance

Colonic inflammation occurs in response to a variety of stimuli and results in smooth muscle dysmotility. Determining the mechanisms of altered protein-protein interaction between ion channels and intracellular signaling molecules are likely to define novel therapeutic approaches targeted at ion channels

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-DKUS-G (09))
Program Officer
Carrington, Jill L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Virginia Commonwealth University
Schools of Medicine
United States
Zip Code
Akbarali, H I; Inkisar, A; Dewey, W L (2014) Site and mechanism of morphine tolerance in the gastrointestinal tract. Neurogastroenterol Motil 26:1361-7
Al-Qudah, M; Anderson, C D; Mahavadi, S et al. (2014) Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C. Am J Physiol Gastrointest Liver Physiol 306:G328-37
AlSharari, Shakir D; Akbarali, Hamid I; Abdullah, Rehab A et al. (2013) Novel insights on the effect of nicotine in a murine colitis model. J Pharmacol Exp Ther 344:207-17
Gade, Aravind R; Kang, Minho; Akbarali, Hamid I (2013) Hydrogen sulfide as an allosteric modulator of ATP-sensitive potassium channels in colonic inflammation. Mol Pharmacol 83:294-306
Hawkins, Edward G; Dewey, William L; Anitha, Mallappa et al. (2013) Electrophysiological characteristics of enteric neurons isolated from the immortomouse. Dig Dis Sci 58:1516-27
Xia, Chun-Mei; Gulick, Melisa A; Yu, Sharon J et al. (2012) Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat. J Neuroinflammation 9:30
Akbarali, H I; G Hawkins, E; Ross, G R et al. (2010) Ion channel remodeling in gastrointestinal inflammation. Neurogastroenterol Motil 22:1045-55
Ross, Gracious R; Gabra, Bichoy H; Dewey, William L et al. (2008) Morphine tolerance in the mouse ileum and colon. J Pharmacol Exp Ther 327:561-72
Kang, Minho; Akbarali, Hamid I (2008) Denitration of L-type calcium channel. FEBS Lett 582:3033-6